恶性疟原虫基因clag9黏附功能研究

发布时间：2019-06-13 18:36

【摘要】： 疟疾是严重危害人类健康的疾病。脑型疟疾(CM)是恶性疟疾的症状之一，也是最主要的致死原因。CM的病理基础是红内期晚期滋养体和裂殖体感染的红细胞与宿主内皮细胞之间的黏附。CM所涉及到的多因素和不同机制迄今尚未阐明。前期文献检索发现，Clag9蛋白是感染红细胞与人脑微血管内皮细胞(HBMVEC)发生黏附的关键蛋白。本实验室前期实验通过生物信息学软件对Clag9进行抗原性和表面可能性分析，选取了Clag9的三个片段Cg1，Cg2，和Cg3，利用原核表达三种多肽片段的GST融合蛋白，检测三种蛋白影响HBMVEC关键的黏附相关受体细胞间黏附分子-1(ICAM-1)表达的情况，以蛋白中检测的对应内毒素剂量做为对照，结果表明，Cg3能够上调宿主细胞受体ICAM-1的表达。但Cg1和Cg2没有类似作用。由于内毒素具有很强的诱导ICAM-1表达的作用，为了更加明确Clag三个片段对于血管内皮细胞黏附功能的影响，必须去除蛋白中的内毒素。本实验的实验策略是：通过构建真核表达载体，，利用哺乳动物细胞表达上述三种Clag9片段或用去除原核表达蛋白中内毒素的方法获得内毒素含量不影响血管内皮细胞表面ICAM-1表达的Clag9片段；用人脐静脉内皮细胞(HUVEC)做为检测蛋白黏附功能的前期实验的静态模型，在体外将蛋白与HUVEC共同孵育，用流式细胞仪检测宿主细胞表面ICAM-1表达的情况。实验结果：去除内毒素后的Cg3与对照组同等剂量的GST对HUVEC的作用结果无显著差异，表明Cg3无明显上调宿主细胞受体ICAM-1表达的作用，结合近期Clag9功能研究的相关文献，Clag9在参与疟原虫感染红细胞与血管内皮细胞黏附方面的功能可能并非通过直接作用于宿主黏附受体而实现，而是通过参与恶性疟感染红细胞表面黏附分子的转运，其功能有待于进一步研究。
[Abstract]:Malaria is a disease that seriously endangers human health. Cerebral malaria (CM) is one of the symptoms of falciparum malaria and the most important cause of death. The pathological basis of CM is the adhesion between erythrocytes and host endothelial cells infected with trophozoites and merozoites in the late erythrocytic phase. The multiple factors and different mechanisms involved in CM have not yet been clarified. It was found that Clag9 protein was the key protein for the adhesion of infected red blood cells to human microvascular endothelial cells (HBMVEC). In our laboratory, the antigenicity and surface possibility of Clag9 were analyzed by bioinformatics software. Three fragments of Clag9, Cg1,Cg2, and Cg3, were selected to express three polypeptide fragments in prokaryotic GST fusion proteins. The effects of three proteins on the expression of HBMVEC key adhesion related receptor cell adhesion molecule-1 (ICAM-1) were detected. The results showed that the corresponding endotoxin dose detected in the protein was used as control. Cg3 can up-regulate the expression of host cell receptor ICAM-1. But Cg1 and Cg2 have no similar effect. Because endotoxin can induce the expression of ICAM-1, in order to clarify the effect of three fragments of Clag on the adhesion function of vascular endothelial cells, it is necessary to remove endotoxin from the protein. The strategy of this experiment is to construct eukaryotic expression vector to express the above three Clag9 fragments by mammal cells or to obtain Clag9 fragments whose endotoxin content does not affect the expression of ICAM-1 on the surface of vascular endothelial cells by removing endotoxin from prokaryotic expression proteins. Human umbilical vein endothelial cell (HUVEC) was used as a static model to detect protein adhesion function. The protein was incubated with HUVEC in vitro, and the expression of ICAM-1 on the surface of host cells was detected by flow cytometry. The results showed that the effect of Cg3 on HUVEC was not significantly different from that of the control group, indicating that Cg3 did not up-regulate the expression of host cell receptor ICAM-1. Combined with the recent research on Clag9 function, the function of Clag9 in the adhesion of red blood cells to vascular endothelial cells infected with Plasmodium falciparum may not be realized by directly acting on the host adhesion receptor. However, by participating in the transport of erythrocyte adhesion molecules in falciparum malaria infection, its function needs to be further studied.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R382

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